In collaboration with clinical investigators in NIDDK, we are continuing our analysis of the antiviral immune response of patients who resolved HCV infection either spontaneously or after interferon-based therapy. Whereas viruses such as HBV, CMV and EBV persist at minute levels after clinical recovery, it is still unclear whether the same is true for hepatitis C virus (HCV). Reports that HCV persists at low levels in individuals who had been diagnosed as having recovered from hepatitis C either spontaneously or after treatment raised significant concerns among patients about the potential consequences of this state of infection. The aim of this study was to test 120 patients at multiple time points 0.5 to 20 years after spontaneous or treatment-induced recovery from HCV-infection for low levels of persisting HCV RNA and for HCV-specific immune responses. Plasma samples of 14/97 (14%) treatment-recovered patients but no spontaneously recovered patients (0/23, 0%) tested positive for trace amounts of HCV RNA in the nested round of 5UTR-specific RT-PCR (sensitivity <40 copies/ml). Linear regression analysis revealed an inverse correlation between the persistence of residual HCV RNA and the time after the end of treatment (R2 = 0.753, p<0.001). Persistence of residual HCV RNA was most frequent in the first years and never observed more than 8 years after the end of treatment. PCR products were sequenced and revealed genotype 1 in one case, genotype 1a/b in 3 cases, genotype 1b in 4 cases, genotype 2b in 3 cases and genotype 3a in one case. The sequences of these PCR products matched the sequence of the dominant pre-treatment HCV in all cases except one. T cell responses of four treatment recovered patients were analyzed at multiple time points after the end of treatment by IFN-? Elispot against 600 overlapping 15mer HCV peptides. In all patients, T cell responses were >20-fold stronger at those time points at which trace amounts of HCV RNA were detected than at time points at which HCV RNA was undetectable. All individuals, for which the persisting HCV RNA sequence matched the pre-treatment HCV sequence, had preserved T cell specificity with responses against nonstructural HCV sequences being dominant. However, for the sole patient for which the persisting HCV RNA sequence (genotype 3) differed from the pre-treatment HCV sequence (genotype 1A), T cell responses changed in specificity from targeting nonstructural HCV sequences at the HCV RNA negative time points to targeting structural HCV sequences at the time point of HCV RNA positivity. In conclusion, trace amounts of HCV RNA may persist at a very low level for an extended but not indefinite time after recovery from hepatitis C. T cell specificity analysis helps to distinguish between viral persistence (preserved immune hierarchy) and new exposure (new immune hierarchy).